The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of 3.8H(+)/2e indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of 3.6H(+)/2e . This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme.     

The Rhodospirillum rubrum cytochrome bc1 complex: redox properties, inhibitor sensitivity and proton pumping.

A detergent-solubilized, three-subunit-containing cytochrome bc1 complex, isolated from the photosynthetic bacterium R. rubrum, has been shown to be highly sensitive to stigmatellin, myxothiazol, antimycin A and UHDBT, four specific inhibitors of these complexes. Oxidation-reduction titrations have allowed the determination of Em values for all the electron-carrying prosthetic groups in the complex. Antimycin A has been shown to produce a red shift in the alpha-band absorbance maximum of one of the cytochrome b hemes in the complex and stigmatellin has been shown to alter both the Em and EPR g-values of the Rieske iron-sulfur protein in the complex. Western blots have revealed antigenic similarities between the cytochrome subunits of the R. rubrum complex and those of the related photosynthetic bacteria, Rb. capsulatus and Rb. sphaeroides. The R. rubrum complex has been incorporated into liposomes. These liposomes exhibit respiratory control and are able to couple electron transfer from quinol to cytochrome c to proton translocation across the liposome membrane in a manner consistent with a Q-cycle mechanism. It can thus be concluded that neither electron transport nor coupled proton translocation by the cytochrome bc1 complex requires more than three subunits in R. rubrum.     

Protein-protein recognition, hydride transfer and proton pumping in the transhydrogenase complex

BACKGROUND: Membrane-bound ion pumps are involved in metabolic regulation, osmoregulation, cell signalling, nerve transmission and energy transduction. How the ion electrochemical gradient interacts with the scalar chemistry and how the catalytic machinery is gated to ensure high coupling efficiency are fundamental to the mechanism of action of such pumps. Transhydrogenase is a conformationally coupled proton pump linking a proton gradient to the redox reaction between NAD(H) and NADP(H). The enzyme has three components; dI binds NAD(H), dII spans the membrane and dIII binds NADP(H). RESULTS: The first crystal structure of a transhydrogenase dI component (from Rhodospirillum rubrum) has been determined at 2.0 A resolution. The monomer comprises two domains. Both are involved in dimer formation, and one has a Rossmann fold that binds NAD+ in a novel mode. The two domains can adopt different conformations. In the most closed conformation, the nicotinamide ring is expelled from the cleft between the two domains and is exposed on the outside of the protein. In this conformation it is possible to dock the structure of dI/NAD+ with that of a dIII/NADP+ complex to provide the first insights into the molecular basis of the hydride-transfer step. CONCLUSIONS: Analysis of the model of the dI/dIII complex identifies residues potentially involved in dI/dIII interaction and shows how domain motion in dI results in a shift in position of the nicotinamide ring of NAD+. We propose that this movement is responsible for switching between the forbidden and allowed states for hydride transfer during proton pumping.     

Allosteric interactions and proton conducting pathways in proton pumping aa3 oxidases: Heme a as a key coupling element

In this paper allosteric interactions in protonmotive heme aa₃ terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H⁺/e^? coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa₃ oxidase, which decreases by more than 200 mV the E_m of heme a, inhibits proton pumping. Mutational aminoacid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa₃ oxidases, as well as Zn^2 + binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O₂ to 2 H₂O. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Refolding and proton pumping activity of a polyethylene glycol-bacteriorhodopsin water-soluble conjugate.

Bacteriorhodopsin (BR), from the purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (m-PEG; molecular weight = 5,000 Da) succinimidyl carbonate. The polyethylene glycol-bacteriorhodopsin (m-PEG-SC-BR33) conjugate, containing one polyethylene glycol chain, was water soluble. The secondary structure of the conjugate in water appeared partially denatured, but was shown to contain alpha-helical segments by circular dichroism spectroscopy. The isolated bacteriorhodopsin conjugate, with added retinal, was refolded in a mixed detergent-lipid micelle and had an absorption maximum at 555 nm. The refolded conjugate was transferred into vesicles that pumped protons, upon illumination, as efficiently as did native BR. Modification of the PM with m-PEG did not alter the native structure or inhibit proton pumping, and therefore it is suggested that the glycol polymer is present as a moiety covalently linked to residues unnecessary for proton pumping and proper folding. The site of attachment of m-PEG was determined to be at either Lys 129 or Lys 159, with position Lys 129 the most probable site of attachment. The m-PEG-SC-BR33 could be stepwise refolded to the native conformation by the addition of trifluoroethanol to lower the dielectric constant, simulating the insertion of the BR into the phospholipid bilayer.     

Formation and implications of a ternary complex of profilin, thymosin beta 4, and actin

Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin beta(4), and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin beta(4) (residues 6-22) confirm the presence of an extensive binding surface between actin and thymosin beta(4), and explain why thymosin beta(4) and profilin can bind simultaneously to actin. Surprisingly, despite much lower affinity, the N-terminal thymosin beta(4) peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurring at the N terminus of thymosin beta(4). Intracellular concentrations of thymosin beta(4) and profilin may greatly exceed the equilibrium dissociation constant of the ternary complex, inconsistent with models showing sequential formation of complexes of profilin-actin or thymosin beta(4)-actin during dynamic remodeling of the actin cytoskeleton. The formation of a ternary complex results in a very large amplification mechanism by which profilin and thymosin beta(4) can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant sequestration even if molecular crowding results in a very low critical concentration of actin in vivo.     

Fromation and stoichiometry of a lysozyme-phospholipid-mitochondrial protein ternary complex.

Mitochondrial membranes reconstituted from lipid-depleted mitochondria and aqueous phospholipid dispersions still have the phospholipid negative charges available for ionic interaction with the basic protein, lysozyme. The stoichiometry of the binding is of about 6 nmoles of lysozyme per 100 nmoles of phospholipid in membranes reconstituted with Asolectin, and of 10 nmoles of phospholipid phosphorus in membranes reconstituted with cardiolipin. Unextracted submitochondrial particles ETP also bind lysozyme (about 3 nmoles per 100 nmoles of phospholipid). These observations indicate that the phospholipid anionic groups are not completely shielded by the mitochondrial proteins, which might occupy areas between the nonpolar groups of the lipid molecules.     

Regions of beta 2 and beta 4 responsible for differences between the steady state dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 neuronal nicotinic receptors

We constructed chimeras of the rat beta 2 and beta 4 neuronal nicotinic subunits to locate the regions that contribute to differences between the acetylcholine (ACh) dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 receptors. Expressed in Xenopus oocytes, the alpha 3 beta 2 receptor displays an EC50 for ACh approximately 20-fold less than the EC50 of the alpha 3 beta 4 receptor. The apparent Hill slope (n(app)) of alpha 3 beta 2 is near one whereas the alpha 3 beta 4 receptor displays an n(app) near two. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other. Exchanging just beta 2:104-120 for the corresponding region of beta 4 shifts the EC50 of ACh dose-response relationship in the expected direction but does not completely convert the EC50 of the dose- response relationship from one wild-type value to the other. However, substitutions in the beta 2:104-120 region do account for the relative sensitivity of the alpha 3 beta 2 receptor to cytisine, tetramethylammonium, and ACh. The expression of beta 4-like (strong) cooperativity requires an extensive region of beta 4 (beta 4:1-301). Relatively short beta 2 substitutions (beta 2:104-120) can reduce cooperativity to beta 2-like values. The results suggest that amino acids within the first 120 residues of beta 2 and the corresponding region of beta 4 contribute to an agonist binding site that bridges the alpha and beta subunits in neuronal nicotinic receptors.     

Allosteric interactions and proton conducting pathways in proton pumping aa(3) oxidases: heme a as a key coupling element

In this paper allosteric interactions in protonmotive heme aa(3) terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H(+)/e(-) coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa(3) oxidase, which decreases by more than 200mV the E(m) of heme a, inhibits proton pumping. Mutational amino acid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa(3) oxidases, as well as Zn(2+) binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O(2) to 2 H(2)O.     

Fluoride inhibition of bovine spleen purple acid phosphatase: characterization of a ternary enzyme-phosphate-fluoride complex as a model for the active enzyme-substrate-hydroxide complex.

Purple acid phosphatases (PAPs) employ a dinuclear Fe(3+)Fe(2+) or Fe(3+)Zn(2+) center to catalyze the hydrolysis of phosphate monoesters. The interaction of fluoride with bovine spleen purple acid phosphatase (BSPAP) has been studied using a combination of steady-state kinetics and spectroscopic methods. For FeZn-BSPAP, the nature of the inhibition changes from noncompetitive at pH 6.5 (K(i(comp)) approximately K(i(uncomp)) approximately 2 mM) to uncompetitive at pH 5.0 (K(i(uncomp)) = 0.2 mM). The inhibition constant for AlZn-BSPAP at pH 5.0 (K(i) = 3 microM) is approximately 50-70-fold lower than that observed for both FeZn-BSAP and GaZn-BSPAP, suggesting that fluoride binds to the trivalent metal. Fluoride binding to the enzyme-substrate complex was found to be remarkably slow; hence, the kinetics of fluoride binding were studied in some detail for FeZn-, AlZn-, and FeFe-BSPAP at pH 5.0 and for FeZn-BSPAP at pH 6.5. Since the enzyme kinetics studies indicated the formation of a ternary enzyme-substrate-fluoride complex, the binding of fluoride to FeZn-BSPAP was studied using optical and EPR spectroscopies, both in the presence and absence of phosphate. The characteristic optical and EPR spectra of FeZn-BSPAP. F and FeZn-BSPAP.PO(4).F are similar at pH 5.0 and pH 6.5, indicating the formation of similar fluoride complexes at both pHs. A structural model for the ternary enzyme-(substrate/phosphate)-fluoride complexes is proposed that can explain the results from both the spectroscopic and the enzyme kinetics experiments. In this model, fluoride binds to the trivalent metal replacing the water/hydroxide ligand that is essential for the hydrolysis reaction to take place, while phosphate or the phosphate ester coordinates to the divalent metal ion.     

The mechanism of cytochrome oxidase and other reaction centres for electron/proton pumping

The functional significance of the metal centres of cytochrome oxidase is deduced from the ways in which the centres are bound into its peptides. To this end use is made of structural knowledge of other metalloproteins for dioxygen binding, haemocyanin and haemoglobin, and for electron transfer, cytochromes b and azurin. The order and manner in which the motions of helical sections of the oxidase are linked to proton pumping are suggested and a comparison is made with other proton pumps, for example that of ATP synthetases.     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a(3) as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK(a) values during the pumping process are derived for comparison to experiments.     

Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. Purification, kinetics, proton pumping, and spectral analysis.

Aerobically grown Rhodobacter sphaeroides synthesizes a respiratory chain similar to that of eukaryotes. We describe the purification of the aa3-type cytochrome c oxidase of Rb. sphaeroides as a highly active (Vmax > or = 1800 s-1), three-subunit enzyme from isolated, washed cytoplasmic membranes by hydroxylapatite chromatography and anion exchange fast protein liquid chromatography. The purified oxidase exhibits biphasic kinetics of oxidation of mammalian cytochrome c, similar to mitochondrial oxidases, and pumps protons efficiently (H+/e- = 0.7) following reconstitution into phospholipid vesicles. A membrane-bound cytochrome c is associated with the aa3-type oxidase in situ, but is removed during purification. The EPR spectra of the Rb. sphaeroides enzyme suggest the presence of a strong hydrogen bond to one or both of the histidine ligands of heme a. In other respects, optical, EPR, and resonance Raman analyses of the metal centers and their protein environments demonstrate a close correspondence between the bacterial enzyme and the structurally more complex bovine cytochrome c oxidase. The results establish this bacterial oxidase as an excellent model system for the mammalian enzyme and provide the basis for site-directed mutational analysis of its energy transducing function.     

The dose-response relationships for myeloid leukemia and malignant lymphoma in BC3F1 mice

The present analysis of data on the induction of lymphoma and myeloid leukemia in BC3F1 mice has indicated some new and interesting aspects regarding the shapes of the dose-effect curves. The incidence data can be interpreted by radiobiological models of the induction process coupled with cell inactivation. In particular, for malignant lymphoma the dose-response curve after X rays can be described assuming a quadratic model corrected for cell inactivation, while the incidence data after fission neutrons are best fitted by a linear model which also allows for cell inactivation. Myeloid leukemia has also been induced in BC3F1 mice. The bell-shaped dose-response curves observed after irradiation with either X rays or neutrons are explained by assuming simultaneous initial transforming events and cell inactivation with the data for cell inactivation at higher doses being in agreement with data reported for other strains of mice. A value for relative biological effectiveness of 4 is obtained at the lowest neutron dose used. The value of the inactivation parameters can be compared with those of the cell inactivation probability per unit dose for the bone marrow hematopoietic stem cells, which are believed to be the target cells for these tumors.     

The quantum efficiency of proton pumping by the purple membrane of Halobacterium halobium.

The quantum yield of H+ release in purple membrane (PM) sheets, and H+ uptake in phospholipid (egg phosphatidylcholine, PC) vesicles containing PM, was measured in single turnover light flashes using a pH-sensitive dye, p-nitrophenol, with rhodopsin as an actinometer. We have also calculated the ratio of H+ released per M412 formed (an unprotonated Shiff-base intermediate formed during the photocycle). In PM sheets, the quantum yield of H+ release depends on the medium. The quantum yield of M412 is independent of salt concentration. The ratio H+/M412 is approximately 1.8 M KC; and approximately 0.64 in 10 mM KCl. Direct measurements of the quantum yield of H+ give approximately 0.7 when the PM is suspended in 0.5 M KC; and 0.25 in 10 mM KCl. Using a quantum yield for M412 formation of 0.3 (Becher and Ebrey, 1977 Biophys J. 17:185.), these measurements also give a H+/M412 approximately 2 at high salt. In PM/PC vesicles, the H+/M412 is approximately 2 at all salt concentrations. The M412 decay is biphasic and the dye absorption change is monophasic. The dissipation of the proton gradient is very slow, taking on the order of seconds. Addition of nigericin (H+/K+ antiporter) drastically reduces the pH changes observed in PM/PC vesicles. This and the observation that the proton relaxation time is much longer than the photochemical cycling time suggest that the protons are pumped across the membrane and there is no contribution as a result of reversible binding and release of protons on just one side of the membrane.     

Challenges in elucidating structure and mechanism of proton pumping NADH:ubiquinone oxidoreductase (complex I)

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the most complicated and least understood enzyme of the respiratory chain. All redox prosthetic groups reside in the peripheral arm of the L-shaped structure. The NADH oxidation domain harbouring the FMN cofactor is connected via a chain of iron–sulfur clusters to the ubiquinone reduction site that is located in a large pocket formed by the PSST- and 49-kDa subunits of complex I. An access path for ubiquinone and different partially overlapping inhibitor binding regions were defined within this pocket by site directed mutagenesis. A combination of biochemical and single particle analysis studies suggests that the ubiquinone reduction site is located well above the membrane domain. Therefore, direct coupling mechanisms seem unlikely and the redox energy must be converted into a conformational change that drives proton pumping across the membrane arm. It is not known which of the subunits and how many are involved in proton translocation. Complex I is a major source of reactive oxygen species (ROS) that are predominantly formed by electron transfer from FMNH₂. Mitochondrial complex I can cycle between active and deactive forms that can be distinguished by the reactivity towards divalent cations and thiol-reactive agents. The physiological role of this phenomenon is yet unclear but it could contribute to the regulation of complex I activity in-vivo.